Replication-defective arenavirus vectors

ABSTRACT

The invention relates to an infectious arenavirus particle that is engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells. One or more of the four arenavirus open reading frames glycoprotein (GP), nucleoprotein (NP), matrix protein Z and RNA-dependent RNA polymerase L are removed or mutated to prevent replication in normal cells but still allowing gene expression in arenavirus vector-infected cells, and foreign genes coding for an antigen or other protein of interest or nucleic acids modulating host gene expression are expressed under control of the arenavirus promoters, internal ribosome entry sites or under control of regulatory elements that can be read by the viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III. The modified arenaviruses are useful as vaccines and therapeutic agents for a variety of diseases.

FIELD OF THE INVENTION

The invention relates to genetically modified arenaviruses suitable asvaccines or gene therapy vectors, and to methods of using these invaccination and treatment of diseases.

BACKGROUND OF THE INVENTION

Preventive vaccines represent one of the most successful chapters ofmodern medicine, having led to the worldwide eradication of smallpox andto the control of polio, measles and many other devastating infectiousdiseases. More recently, vaccines have become available that preventcancer, and strong efforts are ongoing to exploit “vaccines” in atherapeutic fashion, raising hope for both infection and malignancy.Historically, vaccination strategies have comprised a variety ofapproaches: Starting with the use of wild type infectious agents and theauto-(re)-inoculation of tumor cells, followed by live-attenuated agentsand killed tumor tissues, clinical medicine has over time moved more andmore to the use of (inert) proteins and/or other extracts (commonlyreferred to as “antigen”) derived from infectious agents or tumors,respectively. This gradual process represents the search for safervaccine formulation, often accompanied, however, by a relative loss inefficacy. In recent years the advancement of biological engineering hasmade possible yet an additional approach that currently is widelyconsidered among the most promising ones: infectious agents serving as a“ferry” (called “vector”) are equipped with an antigen from the pathogenor tumor of choice. Thereby, the immune response of the vaccinerecipient recognizes the antigen of interest in the context of astrongly immune enhancing (“immunogenic”) context conferred by thevector.

The “vector approach” has also made possible the directed introductionof foreign genes into living cells at the level of tissue culture butalso in multicellular organisms including man, and vectors can thereforealso be exploited for the expression of genes in cultured cells or ingene therapy.

A variety of vectors are currently in experimental use, both forvaccination and gene therapy, with the ultimate goal of optimizingefficacy and safety for clinical application (vaccinology and genetherapy) or for biotechnology (gene transfer in cell culture).

As a common observation, vectors tend to share general traits of theorganism, e.g. virus, they are derived from. The exploitation of a novelfamily of viruses for vector design promises therefore a novelcombination of traits that may confer this new type of vector withunprecedented capabilities and corresponding applications in biomedicalapplication. Vector design needs, however, to take into account thesafety profile of the organism used, and must come up with a strategy ofhow to eliminate the organism's pathogenic potential in a manner thatdoes not interfere with desirable traits such as immunogenicity foradministration as a vaccine.

Arenaviruses in general and lymphocytic choriomeningitis virus (LCMV) inparticular have been known for more than seventy years to elicitextraordinarily strong and long-lasting humoral and cell-mediated immuneresponses. Of note, though, protective neutralizing antibody immunityagainst the viral envelope glycoprotein (GP) is minimal, meaning thatinfection results in minimal antibody-mediated protection againstre-infection if any. Also it has been firmly established for decadesthat owing to their non-cytolytic (not cell-destroying) nature,arenaviruses can, under certain conditions, maintain long-term antigenexpression in animals without eliciting disease. Recently, reversegenetic systems for the manipulation of the infectious arenavirus genome(L. Flatz, A. Bergthaler, J. C. de la Torre, and D. D. Pinschewer, ProcNatl Acad Sci U S A 103:4663-4668, 2006; A. B. Sanchez and J. C. de laTorre, Virology 350:370, 2006) have been described, but arenaviruseshave not so far been exploited as vaccine vectors. Two major obstaclesare mainly responsible: i) Arenaviruses can cause overwhelming infectionwhich then can result in serious disease and immunosuppression. ii) Theincorporation of foreign antigens of choice has not been possible.

SUMMARY OF THE INVENTION

The invention relates to an infectious arenavirus particle that isengineered to contain a genome with the ability to amplify and expressits genetic information in infected cells but unable to produce furtherinfectious progeny particles in normal, not genetically engineeredcells.

More specifically the invention relates to such arenavirus particlescomprising additional ribonucleic acids coding for proteins of interestor modulating host gene expression.

An arenavirus of the invention comprises a modified genome, wherein

i) one or more of the four arenavirus open reading frames glycoprotein(GP), nucleoprotein (NP), matrix protein Z and RNA-dependent RNApolymerase L are removed or mutated to prevent propagation ofinfectivity in normal cells but still allowing gene expression in suchcells;

ii) foreign ribonucleic acids coding for one or more proteins ormodulating host gene expression are introduced and are transcribed fromone or more of the four arenavirus promoters 5′ UTR and 3′ UTR of the Ssegment, and 5′ UTR and 3′ UTR of the L segment, or from additionallyintroduced promoters that can be read by the viral RNA-dependent RNApolymerase, by cellular RNA polymerase I, RNA polymerase II or RNApolymerase III, respectively, and wherein ribonucleic acids coding forproteins or modulating host gene expression are transcribed either bythemselves or as read-through by fusion to arenavirus protein openreading frames; and optionally

iii) one or more internal ribosome entry sites are introduced in theviral transcript sequence to enhance expression of proteins in thearenavirus-infected cell.

The invention furthermore relates to vaccines and pharmaceuticalpreparations comprising such genetically engineered arenaviruses, and tomethods of vaccination and gene therapy using these geneticallyengineered arenaviruses.

The invention furthermore relates to expression of a protein of interestin a cell culture or to modulation of gene expression in cell culturewherein the cell culture is infected with genetically engineeredarenaviruses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Owing to the changes made to the wild type arenavirus genome, thederived arenavirus vectors replicate only in complementing cells.

A: Arenavirus vectors (1) can infect both normal cells (2) orcomplementing cells (C-cells, 3). Upon infection of C-cells furtherinfectious vector progeny are formed whereas infection of normal cellsyields either no vector particles or non-infectious ones.

B: C-cells (1) and normal cells (2) were infected with an LCMV-basedvector (rLCMV/GFP) expressing green fluorescent protein (GFP) instead ofLCMV-GP, and samples of supernatant collected at various time points (3,given in hours). Infectivity in the supernatant (4, given as PFU/ml) wasdetermined in a focus forming assay.

C: The wild type arenavirus genome consists of a large (L; 1) and asmall (S; 2) segment. The L segment expresses the L (3) and Z (4) genes,whereas the S segment carries the NP (5) and GP (6) genes. One strategyto generate a replication-deficient arenavirus vector can be tosubstitute the GP gene for genes of interest, for example GFP (7) orovalbumin (OVA; 8).

FIG. 2 Schematic organization of complementing plasmid (C-plasmid), ofplasmids for intracellular expression of transacting factors(TF-plasmids) and of plasmids for intracellular expression of arenavirusvector genome segments (GS-plasmids).

A: example of a C-plasmid.

B: examples of TF-plasmids expressing the viral NP and L protein,respectively.

C: examples of GS-plasmids expressing the arenavirus vector S and Lsegments, respectively.

1: polymerase II promoter; 2: viral gene to be expressed forcomplementation; 3: internal ribosome entry site; 4: mammalian selectionmarker, such as puromycin resistance gene; 5: polyadenylation signal; 6:ampicillin resistance cassette; 7: origin of replication; 8: viraltrans-acting factor, e.g. NP ORF; 9: viral trans-acting factor, e.g. LORF; 10: promoter driving expression of arenavirus genome segment inC-cells, e.g. polymerase I promoter; 11: 5′ UTR of the S segment; 12:antigen of interest; 13: IGR of the S segment; 14: NP gene; 15: 3′ UTRof the S segment; 16: polymerase I terminator; 17: 5′UTR of the Lsegment; 18: Z gene; 19: IGR of the L segment; 20: L gene; 21: 3′UTR ofthe L segment.

FIG. 3 Arenavirus vectors are cleared within days after inoculation, andaccordingly do not cause immunosuppression in vaccine recipients.

A: On day 0, mice were immunized intravenously with rLCMV/GFP (1). Atdifferent time points thereafter (2; given in days), viral genome copies(3, given in log10) were measured in spleen.

B: As primary immunization/infection (1*), mice were either immunizedwith rLCMV/OVA (5) or were left uninfected (6) or were infected withLCMV wild type (7). On day 20, all mice were given an intraperitonealinfection (2*) with vesicular stomatitis virus (VSV, 8). Subsequently,blood was collected for measuring antiviral and anti-vector T cellresponses (3) and antiviral antibody responses (4). On day 28.H-2K^(b)-SIINFEKL (ovalbumin-derived CD8+ T cell epitope) specific CD8+T cells (9) and H-2K^(b)-VSV-NP52-29 (VSV-NP-derived CD8+ T cellepitope) specific CD8+ T cells (10) were measured in peripheral blood byintracellular staining for interferon gamma upon peptide restimulation(values indicate the percentage of specific cells amongst CD8+ T cells).On day 27 (indicated as “d7” referring to the time point after VSVinfection), day 29 (indicated as “d9”) and day 61 (indicated as “d41”),serum was tested for total VSV neutralizing antibodies (11) and forbeta-mercapto-ethanol-resistant IgG (12) in a 50% plaque reduction assay(values given as −log2 of 40-fold prediluted serum).

FIG. 4 Arenavirus vectors elicit high frequencies of long-lived memoryCD8+ T cells and long-lived antibody memory at high titers.

A: Mice were immunized with rLCMV/OVA and blood samples were collectedover time (2; given in days) for measuring the frequency ofH2K^(b)-OVA/SIINFEKL specific CD8+ T cells using MHC class I tetramers(3, values indicate the frequency of tetramer-positive CD8+ T cellswithin the CD8+ T cell compartment).

B: Mice were immunized with rLCMV/OVA at the indicated dose (1), eithervia the subcutaneous (s.c.) or intravenous (i.v.) route, andOVA-specific IgG in the serum was measured by ELISA on day 14 (5) andday 58 (6). Values are expressed as the dilution of serum yielding twicebackground optical density (OD) measurements.

FIG. 5 Arenavirus vectors do not cause central nervous system disease

Mice were inoculated intracerebrally with rLCMV/OVA (open squares) orwith wild type LCMV (closed circles), and were monitored at theindicated time points (1, indicated in days) for clinical signs ofterminal choriomeningitis. For each time point, the number of healthyanimals per number of animals tested (2) is displayed.

FIG. 6 Arenavirus vectors confer T cell and antibody-mediated protectionagainst infectious challenge.

A) On day 0 of the experiment, mice were vaccinated using eitherrLCMV/OVA (group AA) or using a rLCMV control vector expressing theirrelevant Cre recombinase antigen as negative control (group BB).Intravenous challenge with recombinant Listeria monocytogenes expressingOVA was performed after an interval of either 16 or 58 days (d16, d58).Four days after challenge, bacterial titers (1) were measured in thespleen of the animals (displayed in log₁₀ colony forming units perorgan). Black circles indicate values of individual mice. Vertical linesindicate the mean value of each group.

B) Type I interferon receptor-deficient mice were vaccinated (closedsquares) with a LCMV vector that expresses an antigenic butnon-functional variant of the vesicular stomatitis virus envelopeprotein G (modified by insertion of a foreign peptide sequence in itsectodomain) or were left without vaccination (open circles). One monthlater, all animals were challenged intravenously with 2×10⁶ PFUvesicular stomatitis virus. At the indicated time points after challenge(2, indicated in days), the animals were monitored for clinical signs ofterminal myeloencephalitis. For each time point and group, healthysurvival is indicated as the number of healthy animals per number ofanimals tested (3).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to infectious arenavirus particles, referred to asarenavirus vectors, that are engineered to contain a genome with theability to amplify and express its genetic information in infected cellsbut unable to produce further infectious progeny particles in normal,not genetically engineered cells. This principle is shown schematicallyin FIG. 1A. Example data are presented in FIG. 1B.

Replication of arenavirus vectors requires genetically engineered cellscomplementing the replication-deficient vector. Upon infection of acell, the arenavirus vector genome expresses not only arenavirusproteins but also additional proteins of interest, for example antigensof interest. Arenavirus vectors are produced by standard reverse genetictechniques as described for LCMV (L. Flatz, A. Bergthaler, J. C. de laTorre, and D. D. Pinschewer, Proc Natl Acad Sci U S A 103:4663-4668,2006; B. B. Sanchez and J. C. de la Torre, Virology 350:370, 2006), buttheir genome is modified in one or more of the following ways, resultingin the above-mentioned characteristics:

i) One or more, e.g. two, three or four, of the four arenavirus openreading frames (glycoprotein (GP); nucleoprotein (NP); the matrixprotein Z; the RNA-dependent RNA polymerase L) are removed or mutated toprevent formation of infectious particles in normal cells albeit stillallowing gene expression in arenavirus vector-infected cells.

ii) Foreign nucleic adds coding for one or more proteins can beintroduced. Alternatively or in addition, foreign nucleic acids may beincorporated for modulating host gene expression. These include but arenot limited to short hairpin RNAs (shRNA), small interfering RNA(siRNA), micro RNAs (miRNA), and precursors thereof. These foreignnucleic acids are transcribed from one or more, e.g. two or three of thefour arenavirus promoters 5′ UTR and 3′ UTR of the S segment, and 5′ UTRand 3′ UTR of the L segment, or from additionally introduced promotersequences that can be read by the viral RNA-dependent RNA polymerase, bycellular RNA polymerase I, RNA polymerase II or RNA polymerase III, suchas duplications of viral promoter sequences that are naturally found inthe viral UTRs, the 28S ribosomal RNA promoter, the beta-actin promoteror the 5S ribosomal RNA promoter, respectively. The ribonucleic acidscoding for proteins or modulating host gene expression are transcribedand translated either by themselves or as read-through by fusion toarenavirus protein open reading frames, and expression of proteins inthe host cell may be enhanced by introducing in the viral transcriptsequence at the appropriate place(s) one or more, e.g. two, three orfour, internal ribosome entry sites.

“Modulating host gene expression” as understood herein refers toreduction of expression of host genes or the enhancement thereof, eitherin all vector-targeted cells or in a cell type-specific manner. Thesedesirable features can be achieved by adapting the nucleic acid sequenceincorporated into vectors.

Arenavirus vectors can be used to improve life and health in general,and to immunize (in a preventive manner) or treat (in animmunotherapeutic manner) animals including men in a variety of contextsincluding but not limited to

i) infections including but not limited to viruses such as humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus(HCV), influenza viruses, and respiratory syncytial virus (RSV),bacteria such as mycobacteria, haemophilus spp., and pneumococcus spp.,and parasites such as plasmodia, amebia, and philaria, and prions suchas the infectious agents causing classical and variant Creutzfeldt-Jakobdisease and mad cow disease;

ii) autoimmune diseases including but not limited to type I diabetes,multiple sclerosis, rheumatoid arthritis, lupus erythematosus, andpsoriasis:

iii) neoplastic diseases including but not limited to melanoma, prostatecarcinoma, breast carcinoma, lung carcinoma and neuroblastoma;

iv) metabolic diseases including but not limited to type II diabetes,obesity, and gout;

v) degenerative diseases including but not limited to Alzheimer'sdisease, and Parkinson's disease;

vi) inherited diseases including but not limited to Huntington'sdisease, severe combined immunodeficiency, and lipid storage diseases;

vii) substance dependences including but not limited to tobacco andalcohol abuse; and

viii) allergic diseases including but not limited to seasonal orperennial rhinoconjunctivitis, asthma and eczema.

With the same intention, arenavirus vectors can be used to introduce agene of interest, e.g. foreign nucleic acids, into cells of livinganimals including men, i.e. as gene therapy, or they can be used tointroduce and express a gene product of interest in biotechnologicalapplications. Abolishing replication of arenavirus vectors by deletingfrom their genome e.g. the Z gene which is required for particlerelease, or the GP gene which is required for infection of target cells(compare also FIG. 3), the total number of infected cells is limited bythe inoculum administered, e.g. to a vaccinee or to a recipient of genetherapy, or accidentally transmitted to personnel involved in medical orbiotechnological applications or to animals. Arenavirus disease andimmunosuppression in wild type arenavirus infection are both known toresult from unchecked viral replication. Therefore, abolishingreplication of arenavirus vectors prevents pathogenesis as a result ofintentional or accidental transmission of vector particles. In thisinvention, one important aspect consists in exploiting the abovenecessity of abolishment of replication in a beneficial way for thepurpose of expressing one ore more foreign proteins, e.g. antigens ofinterest: Removal, e.g. structurally by deletion or functionally bymutagenesis, of one or more of the arenavirus genes frees the respectiveviral promoters for expression of the proteins of choice.

A number of combined advantages characterize the present invention onarenavirus vector strategy: Of note, the retained exquisiteimmunogenicity of arenavirus vectors—retained despite the inability ofarenavirus vectors to spread—comes as a great surprise to immunologistsworking in the field of arenavirus immunology. A substantial virus andantigen load over a critical period of time is generally consideredessential for the unmatched immunogenic properties of arenaviruses. Withregard to safety, the virus' (and the vector's) non-cytolytic behavioris a major advantage over most available vector systems, and the sameapplies to the lack of oncogenic potential of arenaviruses in general.Also, the inability of arenavirus vectors to replicate is of muchimportance with regard to safety. Very advantageous, particularly forthe application as vaccines, is also the high level of resistance ofarenavirus vectors to antibody neutralization. This property is inherentto many arenavirus envelopes and allows repeated immunization with thesame arenavirus vector resulting in repeated boosting of the immuneresponse. Similarly, pre-existing immunity against arenaviruses is verylow or negligible in the human population.

Arenaviruses considered are Old World viruses, for example Lassa virus,Lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus,or Ippy virus, or New World viruses, for example Amapari virus, Flexalvirus, Guanarito virus, Junin virus, Latino virus, Machupo virus,Oliveros virus, Paranà virus, Pichinde virus, Pirital virus, Sabiávirus, Tacaribe virus, Tamiami virus, Bear Canyon virus, or WhitewaterArroyo virus. Preferred are members of the Old World viruses, e.g. Lassavirus or LCMV, in particular LCMV.

Foreign nucleic acids coding for one or more proteins of interest aree.g. messenger RNA-derived sequences or RNA corresponding to a primarygene transcript, leading to expression of the protein of interest whenarenavirus particles of the invention carrying this RNA infect a cell.Further foreign nucleic acids considered are those modifying geneexpression in cells infected with the arenavirus vector particle, e.g.by RNA interference.

Ribonucleic acids of interest considered to be introduced in engineeredarenaviruses of the invention are any sequences coding for protein ormodulating host gene expression that can be introduced in an arenavirusvector genome by replacement or fusion to the open reading frame ofglycoprotein GP, the matrix protein Z, the nucleoprotein NP, or thepolymerase protein L, i.e. that can be transcribed and/or expressedunder control of the four arenavirus promoters (5′ UTR and 3′ UTR of theS segment, and 5′ UTR and 3′ UTR of the L segment), as well asribonucleic acids that can be inserted with regulatory elements that canbe read by the viral RNA-dependent RNA polymerase, cellular RNApolymerase I, RNA polymerase II or RNA polymerase III, such asduplications of viral promoter sequences that are naturally found in theviral UTRs, the 28S ribosomal RNA promoter, the beta-actin promoter orthe 5S ribosomal RNA promoter, respectively. The proteins or nucleicacids are transcribed and/or expressed either by themselves or asread-through by fusion to arenavirus open reading frames and genes,respectively, and/or in combination with one or more, e.g. two, three orfour, internal ribosome entry sites. As is demonstrated with genes forGFP and ovalbumin replacing GP, the length of the gene inserted and theproperties of the expressed protein are not critical and open thepossibility for expression of a large variety of proteins of interest.

Preferred proteins of interest are peptidic or proteinaceous antigens.Peptidic or proteinaceous antigens of the invention may, for example, beselected from the group consisting of (a) proteins or peptides suited toinduce or modulate an immune response against infectious diseases; (b)proteins or peptides suited to induce or modulate an immune responseagainst neoplastic diseases, i.e. cancer cells; and (c) proteins orpeptides suited to induce or modulate an immune response againstallergens. Combinations of antigens, e.g. of antigens derived from oneor more infectious organisms or tumors or allergens may be combined toelicit or modulate an immune response protecting or curing more than oneinfection, type of tumor or allergic disease, respectively.

“Modulating an immune response” as used herein means i) improving,either in quality or quantity, a beneficial immune response of apatient. This is desirable, for example, when enhancing HIV-specific Tcell and antibody responses in the context of immunotherapy of aninfected individual. The term “modulating an immune response” alsorefers to ii) the process generally known as desensitization, e.g.desensitization against allergens by suppressing an allergic type ofimmune response such as the one of the immunoglobulin E isotype, withthe attempt of substituting or superimposing a protective immuneresponse or of dampening the pathogenic immune response.

In one specific embodiment of the invention, the antigen is one that isuseful for the prevention of infectious disease. Particular examples ofantigens or antigenic determinants include the HIV antigens gp 41, gp120, gag, and pol, non-structural (NS) proteins of Hepatitis C virus,the influenza antigens hemagglutinin and neuraminidase, hepatitis Bsurface antigen, and circumsporozoite protein of malaria.

Preferably the antigen is selected from respiratory syncytial virusantigens, human immunodeficiency virus antigens, hepatitis C virusantigens, varicella zoster virus antigens, herpes simplex virusantigens, cytomegalovirus antigens and antigens derived frommycobacterium tuberculosis.

The selection of antigens for the composition and method of treatmentfor cancer would be known to those skilled in the medical art treatingsuch disorders. Representative examples of this type of antigen includethe following: HER2/neu (breast cancer). GD2 (neuroblastoma), EGF-R(malignant glioblastoma), CEA (medullary thyroid cancer), CD52(leukemia), MUC1 (expressed in hematological malignancies), gp100protein, MELAN-A/MART1 or the product of the tumor suppressor gene WT1.

The selection of antigens for the composition and method of treatmentfor allergy would be known to those skilled in the medical art treatingsuch disorders. Representative examples of this type of antigen includebut are not limited to birch pollen antigen Bet v 1 and cat allergen Feld 1.

The selection of antigens for the composition and method of treatmentfor obesity would be known to those skilled in the medical art treatingsuch disorders. Representative examples of this type of antigen includebut are not limited to ghrelin and gastric inhibitory peptide (GIP).

Design of Arenavirus Vector Genome

Starting out from a wild type arenavirus genome (FIG. 1C), thearenavirus vector genome is designed to retain at least the essentialregulatory elements on the 5′ and 3′ untranslated regions (UTRs) of bothsegments, and preferentially also the intergenic regions (IGRs). Theminimal transacting factors necessary for gene expression in infectedcells remain in the vector genome as open reading frames that can beexpressed, yet they can be placed differently in the genome and can beplaced under control of a different promoter than naturally, or can beexpressed from internal ribosome entry sites. At least one of the fourviral genes (NP, L, GP, Z) is removed or is functionally inactivated.One or more additional genes or stretches of nucleic acids of interestare inserted into the arenavirus vector genome, placed and oriented toallow their expression in infected cells, either under control of one ofthe four viral promoters (5′ and 3′ UTR of the S segment, 5′ and 3′ UTRof the L segment) or from an internal ribosome entry site or frompromoters that can be read by the viral RNA-dependent RNA polymerase,cellular RNA polymerase I, RNA polymerase II or RNA polymerase III. FIG.1C shows one example where the arenavirus GP open reading frame (ORF) isreplaced by either an ovalbumin (OVA) or green fluorescent protein (GFP)ORF.

Generation of a Complementing Cell Line

Owing to the “deletion” (referring to either removal or functionalinactivation) of one or more of the viral genes in arenavirus vectors(here deletion of the glycoprotein, GP, will be taken as an example),arenavirus vectors must be generated and expanded on cells providing intrans the deleted viral gene(s), e.g. the GP in the present example.Such a complementing cell line, henceforth referred to as C-cells, isgenerated by transfecting a mammalian cell line such as BHK-21, HEK293,VERO or other (here BHK-21 will be taken as an example) with one or moreplasmid(s) for expression of the viral gene(s) of interest(complementation plasmid, referred to as C-plasmid). The C-plasmid(s)(for an example see FIG. 2A) express the viral gene(s) deleted in thearenavirus vector to be generated under control of one or moreexpression cassettes suitable for expression in mammalian cells, e.g. amammalian polymerase II promoter such as the CMV or EF1alpha promoterwith a polyadenylation signal. In addition, the complementation plasmidfeatures a mammalian selection marker, e.g. puromycin resistance, undercontrol of an expression cassette suitable for gene expression inmammalian cells, e.g. polymerase II expression cassette as above, or theviral gene transcript(s) are followed by an internal ribosome entrysite, such as the one of encephalomyocarditis virus, followed by themammalian resistance marker. For production in E. coli, the plasmidadditionally features a bacterial selection marker, such as anampicillin resistance cassette.

The cells to be used, e.g. BHK-21, HEK293, MC57G or other, are kept inculture and are transfected with the complementation plasmid(s) usingany of the commonly used strategies such as calcium-phosphate-,liposome-based protocols or electroporation. A few days later thesuitable selection agent, e.g. puromycin, is added in titratedconcentrations. Surviving clones are isolated and subcloned followingstandard procedures, and high-expressing C-cell clones are identifiedusing Western blot or flow cytometry procedures with antibodies directedagainst the viral protein(s) of interest. As an alternative to the useof stably transfected C-cells transient transfection of normal cells cancomplement the missing viral gene(s) in each of the steps where C-cellswill be used below.

Plasmids for the Recovery of Arenavirus Vectors

Plasmids needed are of two types:

i) Two plasmids, referred to as TF-plasmids (for an example see FIG.2B), for expressing intracellularly in C-cells the minimal transactingfactors of the arenavirus the vector is derived from e.g. NP and Lproteins of LCMV in the present example.

ii) Plasmids, referred to as GS-plasmids (for an example see FIG. 2C),for expressing intracellularly in C-cells the arenavirus vector genomesegments, e.g. the segments with designed modifications as described inFIG. 1C. TF-plasmids express the NP and L proteins of the respectivearenavirus vector under control of an expression cassette suitable forprotein expression in mammalian cells, typically e.g. a mammalianpolymerase II promoter such as the CMV or EF1alpha promoter, either oneof them preferentially in combination with a polyadenylation signal(FIG. 2B). GS-plasmids express the small (S) and the large (L) genomesegments of the vector. Typically, polymerase I-driven expressioncassettes (FIG. 2C) or T7 bacteriophage RNA polymerase (T7-) drivenexpression cassettes can be used, the latter preferentially with a3′-terminal ribozyme for processing of the primary transcript to yieldthe correct end. In the case of using a T7-based system, expression ofT7 in C-cells must be provided by either including in the recoveryprocess an additional expression plasmid, constructed analogously toTF-plasmids, providing T7, or C-cells are constructed to additionallyexpress T7 in a stable manner.

Recovery of the Arenavirus Vector

First day: C-cells, typically 80% confluent in M6-well plates, aretransfected with a mixture of the two TF-plasmids plus the twoGS-plasmids. For this one can exploit any of the commonly usedstrategies such as calcium-phosphate-, liposome-based protocols orelectroporation.

3-5 days later: The culture supernatant (arenavirus vector preparation)is harvested, aliquoted and stored at 4° C., −20° C. or −80° C.depending on how long the arenavirus vector should be stored prior touse. Then the arenavirus vector preparation's infectious titer isassessed by an immunofocus assay on C-cells.

Titration of Arenavirus Vector Infectivity

For measuring the infectivity of an arenavirus vector preparationC-cells are used for a typical immunofocus assay following commonly usedprinciples in virology as outlined hereinafter:

C-cell monolayers, typically in M24 well plates, 80% confluent, areinfected with 10-fold dilutions of the arenavirus vector preparation for90 min. Subsequently, the cell layer is overlayed with suitable cellculture medium supplemented with 1% methylcellulose. Two to three dayslater, depending on the permissiveness of the C-cell line used, theculture supernatant is removed, the cell layer is fixed, typically withethanol/acetone or with formalin 4%, followed by permeabilization of thecell layer using mild detergents. Subsequently,arenavirus-vector-infected cell foci are identified using mono- orpolyclonal antibody preparation(s) against one of the proteins in thearenavirus vector to be tested or against the antigen introduced. Boundantibody is detected using appropriate reagents, such as anti-isotype oranti-species antibodies that are conjugated to a system forvisualization such as horse radish peroxidase, followed by a colorreaction with suitable chromogens such as o-phenylenediamine. Theresulting spots on the plate are counted to calculate the number ofinfectious focus forming units (FFU) per volume of arenavirus vectorpreparation.

Vaccines and Pharmaceutical Preparations

The invention furthermore relates to vaccines and pharmaceuticalpreparations comprising the genetically engineered arenaviruses asdescribed hereinbefore. Vaccines and pharmaceutical preparations forother uses are prepared according to standard procedures in the art.

Compositions for enteral administration, such as nasal, buccal, rectalor oral administration, and for parenteral administration, such asintravenous, intramuscular, intradermal or subcutaneous administration,to warm-blooded animals, especially humans, are preferred. Particularlypreferred are compositions for parenteral administration. Thecompositions comprise the genetically engineered arenaviruses alone or,preferably, together with a pharmaceutically acceptable carrier. Thedosage of the active ingredient depends upon the type of vaccination andthe disease to be treated and upon the species, its age, weight, andindividual condition, the individual pharmacokinetic data, and the modeof administration.

The pharmaceutical compositions comprise from about 10³ to about 10¹¹focus forming units of the genetically engineered arenaviruses. Unitdose forms for parenteral administration are, for example, ampoules orvials, e.g. vials containing from about 10³ to 10¹⁰ focus forming unitsor 10⁵ to 10¹⁵ physical particles of genetically engineeredarenaviruses.

Preference is given to the use of suspensions or dispersions ofgenetically engineered arenaviruses, especially isotonic aqueousdispersions or suspensions. The pharmaceutical compositions may besterilized and/or may comprise excipients, e.g. preservatives,stabilizers, wetting agents and/or emulsifiers, solubilizers, salts forregulating osmotic pressure and/or buffers and are prepared in a mannerknown per se, for example by means of conventional dispersing andsuspending processes. The said dispersions or suspensions may compriseviscosity-regulating agents. The suspensions or dispersions are kept attemperatures around 2-4° C., or preferentially for longer storage may befrozen and then thawed shortly before use.

The invention relates also to processes and to the use of geneticallyengineered arenaviruses for the manufacture of vaccines in the form ofpharmaceutical preparations, which comprise genetically engineeredarenaviruses as active ingredient. The pharmaceutical compositions ofthe present invention are prepared in a manner known per se, for exampleby means of conventional mixing and/or dispersing processes.

Administration to Vaccinee and to Gene Therapy Recipient

The invention furthermore relates to methods of vaccination and genetherapy using the genetically engineered arenaviruses as describedhereinbefore.

Arenavirus vectors are administered for improving the quality of live,including but not limited to vaccination, immunotherapy and gene therapyin order to prevent, treat or improve

i) infections including but not limited to those caused by viruses suchas human immunodeficiency virus (HIV), hepatitis B virus (HBV),hepatitis C virus (HCV), influenza viruses, and respiratory syncytialvirus (RSV), bacteria such as mycobacteria, haemophilus spp., andpneumococcus spp., parasites such as plasmodia, amebia, and philaria,and prions such as the infectious agents causing classical and variantCreutzfeldt-Jakob disease and mad cow disease;

ii) autoimmune diseases including but not limited to type I diabetes,multiple scierosis, rheumatoid arthritis, lupus erythematosus, andpsoriasis;

iii) neoplastic diseases including but not limited to melanoma, prostatecarcinoma, breast carcinoma, lung carcinoma, and neuroblastoma;

iv) metabolic diseases including but not limited to type II diabetes,obesity, and gout;

v) degenerative diseases including but not limited to Alzheimer'sdisease and Parkinson's disease;

vi) inherited diseases including but not limited to Huntington'sdisease, severe combined immunodeficiency, and lipid storage diseases;and

vii) substance dependence including but not limited to tobacco andalcohol abuse.

In particular the invention relates to a method of preventing infectionsby viruses, bacteria, parasites and prions comprising administering avaccine comprising genetically engineered arenaviruses to a patient inneed thereof, and likewise to a method of preventing neoplastic diseasesand degenerative diseases as listed hereinbefore.

Furthermore the invention relates to a method of treating infections byviruses, bacteria, parasites and prions, autoimmune diseases, neoplasticdiseases, metabolic diseases, degenerative diseases, inherited diseases,or substance dependence comprising administering a pharmaceuticalpreparation comprising genetically engineered arenaviruses to a patientin need thereof.

Arenavirus vectors are administered to a vaccinee either by one or bymultiple ones of the available routes including but not limited tointramuscular, intradermal, subcutaneous, peroral, intranasal, orintravenous routes, e.g. as in the experiment outlined in FIG. 3A. Thisresults in infection of cells, amplification of the viral genomesegments in these very same initially infected cells, e.g. afterintravenous inoculation. This comprises dendritic cells of the spleenthat can trigger T cell responses. Owing to the inability of thearenavirus vector to replicate in cells of a vaccinee, lacking thecomplementing viral protein present in C-cells, the levels of arenavirusvector RNA decline rapidly over time and the viral genome approaches itsextinction within days after inoculation of the arenavirus vector (FIG.3A). Owing to the lack of arenavirus vector replication and persistence,and in contrast to infection with the same dose of wild type virus,arenavirus vector immunization does not cause immunosuppression (FIG.3B) or disease (FIG. 5). This is tested in mice infected either withwild type LCMV or with an LCMV-based vector expressing OVA instead ofLCMV-GP (rLCMV/OVA; compare FIG. 1C). Subsequent infection withvesicular stomatitis virus elicited a normal CD8 T cell and antibodyresponse in animals previously immunized with rLCMV/OVA, but wassuppressed in animals previously infected with wild type LCMV.Similarly, wild type LCMV elicited lethal choriomeningitis in mice whenadministered intracranially, but rLCMV/OVA did not elicit any clinicallydetectable signs of illness (FIG. 5).

Despite its transient nature, expression of the antigen of interestdoes, however, evoke a strong and long-lasting T cell response (FIG. 4A)and evokes high titers of specific antibodies (FIG. 4B). This responseis dose-dependent but even small doses are efficient (FIG. 4B). Theprotective capacity of T cell responses elicited by LCMV-based vaccinevectors is tested in mice. Immunization with rLCMV/OVA protected againstinfectious challenge with recombinant Listeria monocytogenes expressingOVA (rLM/OVA). This was evident in strongly reduced or undetectablerLM/OVA titers in the spleen of vaccinated animals (FIG. 6A). Inductionof antibody-mediated protection by LCMV vector is tested in mice lackingthe type I interferon receptor. These mice are highly susceptible tovesicular stomatitis virus (VSV), with a 50% lethal dose (LD₅₀) in therange of 50 PFU. For immunization against VSV, a LCMV vector was usedthat expresses an antigenic but non-functional variant of the vesicularstomatitis virus envelope protein G (modified by insertion of a foreignpeptide sequence in its ectodomain). Immunized mice survived a challengeinfection with 2×10⁶ PFU VSV (i.e. >10,000-fold LD₅₀), whereasunimmunized control mice developed terminal myeloencephalitis within twoto three days after VSV challenge (FIG. 6). Of note, though,inactivation of the arenavirus vector genome by UV irradiation abrogatedits immunogenicity, demonstrating that replication and gene expressionof the viral vector in infected cells is essential for its efficacy as avaccine. Additionally, T cell and antibody responses can be enhanced byrepeated applications of the same (homologous) or different(heterologous) arenavirus vectors, i.e. in the form of boosterimmunization. In homologous prime-boost regimens, the virtual absence ofneutralizing antibody induction renders booster immunizationsparticularly efficient.

When used for gene therapy, arenavirus vectors can be appliedsystemically, e.g. intravenously, or topically, e.g. by stereotacticinjection using appropriate equipment, for targeting and delivery tospecific tissues where the antigen of interest should be expressed.Owing to its non-cytolytic nature, the arenavirus vector does not harmthe cell it infects and can functionally substitute for a gene ofinterest.

As an alternative way of exploiting arenavirus vectors for treatment ofmulticellular organisms, complementing (C-cells) or non-complementing(normal) cells are implanted into a recipient's body encapsulated bybiocompatible materials preventing immune rejection by the recipient yetallowing for the constant release of infectious (implantation ofinfectious C-cells) or non-infectious (implantation of infected normalcells) particles or of proteins and/or ribonucleic acids from theencapsulated cells across the capsule into the recipient's tissues.

Expression of a Protein of Interest in a Cell Culture

The invention furthermore relates to expression of a protein of interestin a cell culture wherein the cell culture is infected with geneticallyengineered arenaviruses. When used for expression of a protein orstretch of nucleic acids of interest, e.g. an antigen of interest, incultured cells, the following two procedures are envisaged:

i) The cell type of interest is infected with the arenavirus vectorpreparation at a multiplicity of infection (MOI) of one or more, e.g.two, three or four, resulting in production of the protein of interestin all cells already shortly after infection.

ii) Alternatively, a lower MOI can be used and individual cell clonescan be selected for their level of virally driven protein expression.Subsequently individual clones can be expanded infinitely owing to thenon-cytolytic nature of arenavirus vectors. Irrespective of theapproach, the protein(s) of interest can subsequently be collected (andpurified) either from the culture supernatant or from the cellsthemselves, depending on the properties of the protein(s) produced.

However, the invention is not limited to these two strategies, and otherways of driving expression of proteins or nucleic acids of interestusing genetically engineered arenaviruses as vectors may be considered.

1. An infectious arenavirus particle engineered to contain a genome withthe ability to amplify and express its genetic information in infectedcells but unable to produce further infectious progeny particles innormal, not genetically engineered cells.
 2. The arenavirus particleaccording to claim 1 comprising additional nucleic acids coding for aprotein or peptide of interest.
 3. The arenavirus particle according toclaim 1 comprising additional nucleic acids modulating host geneexpression.
 4. The arenavirus particle according to claim 2 comprising amodified genome, wherein i) one or more of the four arenavirus openreading frames glycoprotein (GP), nucleoprotein (NP), matrix protein Zand RNA-dependent RNA polymerase L are removed or mutated to preventreplication in normal cells but still allowing gene expression inarenavirus vector-infected cells; ii) foreign ribonucleic acids codingfor one or more proteins of interest are expressed under control of oneor more of the four arenavirus promoters 5′ UTR and 3′ UTR of the Ssegment, and 5′ UTR and 3′ UTR of the L segment, and/or under control ofregulatory elements that can be read by the viral RNA-dependent RNApolymerase, cellular RNA polymerase I, RNA polymerase II or RNApolymerase III, expressed either by themselves or as read-through byfusion to arenavirus protein open reading frames, and optionally iii)one or more internal ribosome entry sites are introduced to enhanceexpression of proteins of interest in the arenavirus vector-infectedcell.
 5. The arenavirus particle according to claim 4 wherein thearenavirus open reading frame glycoprotein (GP) is removed or mutated.6. The arenavirus particle according to claim 1 wherein the arenavirusopen reading frame glycoprotein (GP) of the genome is removed andreplaced by foreign ribonucleic acids coding for one or more proteins ofinterest.
 7. The arenavirus particle according to claim 1 wherein thearenavirus open reading frame glycoprotein (GP) of the genome is removedand replaced by foreign ribonucleic acids coding for peptide or proteinantigens derived from infectious organisms, tumors or allergens.
 8. Thearenavirus particle according to claim 1 wherein the arenavirus openreading frame glycoprotein (GP) of the genome is removed and replaced byforeign ribonucleic acids selected from short hairpin RNAs (shRNA),small interfering RNA (siRNA) and micro RNAs (miRNA).
 9. The arenavirusparticle according to claim 1 wherein the arenavirus is lymphocyticchoriomeningitis virus (LCMV).
 10. The arenavirus particle according toclaim 9 comprising foreign ribonucleic acids coding for an antigenselected from respiratory syncytial virus antigens, humanimmunodeficiency virus antigens, hepatitis C virus antigens, varizellazoster virus antigens, herpes simplex virus antigens, cytomegalovirusantigens and antigens derived from mycobacterium tuberculosis.
 11. Avaccine or pharmaceutical preparation comprising an arenavirus particleaccording to claim
 2. 12. A method of preventing infections by viruses,bacteria, parasites and prions in a patient comprising administering atherapeutically effective amount of an arenavirus particle according toclaim 2 to a patient in need thereof.
 13. A method of treatinginfections caused by viruses, bacteria, parasites and prions, autoimmunediseases, neoplastic diseases, metabolic diseases, degenerativediseases, inherited diseases, allergic diseases, or substance dependencein a patient comprising administering a therapeutically effective amountof an arenavirus particle according to claim 2 to a patient in needthereof.
 14. A method of expressing a protein of interest in a cellculture wherein the cell culture is infected with an arenavirus particleaccording to claim
 2. 15. A plasmid for expressing an arenavirus genomesegment comprising a nucleic acid sequence that can express anarenavirus genome segment, wherein the arenavirus genome segment is an Ssegment or an L segment, and wherein an arenavirus open reading frame ofthe arenavirus genome segment that encodes a glycoprotein (GP), anucleoprotein (NP), a matrix protein Z or a RNA-dependent RNA polymeraseis removed or functionally inactivated.
 16. The plasmid of claim 15,wherein an arenavirus particle comprising the arenavirus genome segmentis infectious, but unable to produce further infectious progenyparticles in non-complementing cells, and wherein the genome of thearenavirus particle is engineered to have the ability to amplify andexpress its genetic information in arenavirus infected cells.
 17. Theplasmid of claim 15, wherein the arenavirus genome segment is an Ssegment and the arenavirus open reading frame of the arenavirus genomesegment that encodes the GP is removed or functionally inactivated. 18.The plasmid of claim 17, wherein the GP is removed and replaced with aforeign ribonucleic acid sequence encoding protein or peptide ofinterest.
 19. A complementing cell for the production of an arenavirusparticle, wherein the complementing cell comprises a plasmid of claim15, and expresses the open reading frame that has been removed orfunctionally inactivated from the arenavirus genome.
 20. A method forproducing replication-deficient infectious arenavirus particlescomprising culturing the complementing cell of claim 19 and harvestingthe culture supernatant comprising said arenavirus particles.